Computational Structure Models of Apo and Diferric Transferrin–Transferrin Receptor Complexes

Treatment of K562 cells with desferrioxamine, a permeable iron chelator, led to an increase in the number of transferrin receptors. Increasing intracellular iron levels by treatment of cells with either human diferric transferrin or hemin lowered the level of the transferrin receptors. By using a cDNA clone of the human transferrin receptor, we showed that the changes in the levels of the receptor by iron were accompanied by alterations in the levels of the mRNA for the receptor. The rapidity of these changes indicated that the mRNA had a very short half-life. By using an in vitro transcriptional assay with isolated nuclei, we obtained evidence that this regulation occurred at the transcriptional level.

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Effect of iron on transferrin receptor expression by human placental syncytiotrophoblast cells

Reproduction Fertility and Development ◽

10.1071/r97021 ◽

1997 ◽

Vol 9 (6) ◽

pp. 609 ◽

Cited By ~ 3

Author(s):

Martha L. Kennedy ◽

Gordon C. Douglas ◽

Barry F. King

Keyword(s):

Transferrin Receptor ◽

Iron Status ◽

Primary Cultures ◽

Receptor Expression ◽

Transferrin Receptors ◽

Iron Salts ◽

Intracellular Receptor ◽

Cytotrophoblast Cells ◽

Diferric Transferrin ◽

Transferrin Receptor Expression

Transferrin receptor expression has been examined in primary cultures of morphologically differentiated placental syncytiotrophoblast cells. More than 90% of the cells were multinucleated. Incubation of syncytiotrophoblast for 4 days in the presence of iron salts had no effect on receptor expression assessed by measuring the binding of 125I-labelled transferrin. However, incubation of cells in the presence of human diferric transferrin (10-100 µM) led to a 50% decrease in surface and intracellular receptor expression. This down-regulation was not accompanied by a signicant decrease in receptor synthesis. In contrast to syncytiotrophoblast, expression of intracellular transferrin receptors in non-differentiated cytotrophoblast cells decreased when cells were cultured with iron salts; this was accompanied by decreased receptor synthesis. Addition of diferric transferrin to cytotrophoblast cells led to a 50% reduction in surface and intracellular receptor expression, similar to that seen in the syncytiotrophoblast. This reduction was accompanied by a decrease in receptor synthesis. In contrast to that of most cell types, the expression and distribution of trophoblast transferrin receptors were not altered by insulin, epidermal growth factor or hydrocortisone. These characteristics of syncytiotrophoblast transferrin receptor expression may assist in ensuring a supply of iron to the fetus regardless of the maternal iron status.

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Isolation of an endocytic compartment from A431 cells using a density modification procedure employing a receptor-specific monoclonal antibody complexed with colloidal gold

Journal of Cell Science ◽

10.1242/jcs.87.4.495 ◽

1987 ◽

Vol 87 (4) ◽

pp. 495-506

Author(s):

J. Beardmore ◽

K.E. Howell ◽

K. Miller ◽

C.R. Hopkins

Keyword(s):

Transferrin Receptor ◽

Colloidal Gold ◽

A431 Cells ◽

Receptor Complexes ◽

Fold Enrichment ◽

Versus Protein ◽

Continuous Sucrose Gradient ◽

Modification Procedure ◽

Enzyme Markers ◽

Human Epidermoid Carcinoma

Our objective was to isolate a prelysosomal compartment involved in receptor-mediated endocytosis in human epidermoid carcinoma (A431) cells. The isolation protocol involves density modification of endosome elements in A431 cells, caused by the receptor-dependent binding and internalization at 20 degrees C of colloidal gold-transferrin receptor antibody (B3/25) particles. The use of 125I-labelled gold-B3/25 provides a radioactive marker for the endosome compartment, the major peak being recovered at the bottom of a continuous sucrose gradient at a density of 1.23g ml-1. Enzyme markers characteristic of other cytoplasmic compartments are present only in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicates approximately a 200-fold enrichment of 125I-labelled gold-B3/25 versus protein. Electron microscopy of the endosome-rich fraction reveals that we have isolated a highly purified population of small gold-containing vesicles and tubules from which the transferrin receptor can be immunoprecipitated using the B3/25 antibody. Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that these elements contain a characteristic profile of approximately 10 major proteins of which three appear to be specifically enriched. In cells incubated with [125I]transferrin, 12% of the ligand sediments with the gold-labelled elements. We conclude, therefore, that the components we have isolated play a role in the intracellular processing of the transferrin-transferrin receptor complexes.

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The rate of internalization of different receptor–ligand complexes in alveolar macrophages is receptor-specific

Biochemical Journal ◽

10.1042/bj2700369 ◽

1990 ◽

Vol 270 (2) ◽

pp. 369-374 ◽

Cited By ~ 9

Author(s):

D M Ward ◽

J Kaplan

Keyword(s):

Alveolar Macrophages ◽

Receptor Occupancy ◽

Receptor Internalization ◽

Coated Pits ◽

Surface Receptors ◽

Receptor Complexes ◽

Rate Limiting Step ◽

Internalization Rate ◽

The Absolute ◽

Diferric Transferrin

To probe the mechanisms of endocytosis in alveolar macrophages, we examined the internalization rates of three different receptors. Initial rates of internalization for mannosylated BSA, diferric transferrin and alpha-macroglobulin-proteinase complexes were all different. Although the absolute rates of internalization varied depending on the cell preparation, transferrin was internalized at 10-20% and alpha-macroglobulin-proteinase complex at 40-60% of the rate of manosylated-BSA. Incubation of cells with transferrin did not affect the rate of internalization of mannosylated BSA or alpha-macroglobulin-proteinase complexes, and the rates of internalization were independent of receptor occupancy. These different internalization rates could not be ascribed to different rates of diacytosis. Altering the distribution of unoccupied surface receptors by either trypsin treatment of cells at 0 degree C or exposure to hyperosmotic solutions resulted in the absolute internalization rates being affected by the experimental condition, but the hierarchy in receptor internalization rates was maintained. The fact that a variety of conditions affect receptor internalization rates to the same degree implies the existence of co-ordinate regulation at a single rate-limiting step. Based on these results, we suggest that differences in internalization rate reflect the ability of ligand-receptor complexes to be captured by coated pits.

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Diferric transferrin regulates transferrin receptor 2 protein stability

Blood ◽

10.1182/blood-2004-06-2477 ◽

2004 ◽

Vol 104 (13) ◽

pp. 4287-4293 ◽

Cited By ~ 163

Author(s):

Martha B. Johnson ◽

Caroline A. Enns

Keyword(s):

Transferrin Receptor ◽

Iron Homeostasis ◽

Transmembrane Protein ◽

Hereditary Hemochromatosis ◽

Pcr Analysis ◽

Mrna Levels ◽

Dependent Manner ◽

Diferric Transferrin ◽

In Cells ◽

Transferrin Receptor 2

Abstract Transferrin receptor 2 (TfR2) is a type 2 transmembrane protein expressed in hepatocytes that binds iron-bound transferrin (Tf). Mutations in TfR2 cause one form of hereditary hemochromatosis, a disease in which excessive absorption of dietary iron can lead to liver cirrhosis, diabetes, arthritis, and heart failure. The function of TfR2 in iron homeostasis is unknown. We have studied the regulation of TfR2 in HepG2 cells. Western blot analysis shows that TfR2 increases in a time- and dose-dependent manner after diferric Tf is added to the culture medium. In cells exposed to diferric Tf, the amount of TfR2 returns to control levels within 8 hours after the removal of diferric Tf from the medium. However, TfR2 does not increase when non–Tf-bound iron (FeNTA) or apo Tf is added to the medium. The response to diferric Tf appears to be hepatocyte specific. Real-time quantitative reverse transcription–polymerase chain reaction (qRT-PCR) analysis shows that TfR2 mRNA levels do not change in cells exposed to diferric Tf. Rather, the increase in TfR2 is attributed to an increase in the half-life of TfR2 protein in cells exposed to diferric Tf. Our results support a role for TfR2 in monitoring iron levels by sensing changes in the concentration of diferric Tf.

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Comparison of the kinetics of cycling of the transferrin receptor in the presence or absence of bound diferric transferrin

Biochemical Journal ◽

10.1042/bj2640035 ◽

1989 ◽

Vol 264 (1) ◽

pp. 35-46 ◽

Cited By ~ 31

Author(s):

N Gironès ◽

R J Davis

Keyword(s):

Rate Constants ◽

Transferrin Receptor ◽

Defined Medium ◽

Carcinoma Cells ◽

Endosomal Membrane ◽

Fab Fragments ◽

Membrane Compartments ◽

Diferric Transferrin ◽

Kinetics Of ◽

Human Epidermoid Carcinoma

The kinetics of cycling of the transferrin receptor in A431 human epidermoid-carcinoma cells was examined in the presence or absence of bound diferric transferrin. In order to investigate the properties of the receptor in the absence of transferrin, the cells were maintained in defined medium without transferrin. It was demonstrated that Fab fragments of a monoclonal anti-(transferrin receptor) antibody (OKT9) did not alter the binding of diferric 125I-transferrin to the receptor or change the accumulation of [59Fe]diferric transferrin by cells. OKT9 125I-Fab fragments were prepared and used as a probe for the function of the receptor. The first-order rate constants for endocytosis (0.16 +/- 0.02 min-1) and exocytosis (0.056 +/- 0.003 min-1) were found to be significantly lower for control cells than the corresponding rate constants for endocytosis (0.22 +/- 0.02 min-1) and exocytosis (0.065 +/- 0.004 min-1) measured for cells incubated with 1 microM-diferric transferrin (mean +/- S.D., n = 3). The cycling of the transferrin receptor is therefore regulated by diferric transferrin via an increase in both the rate of endocytosis and exocytosis. Examination of the accumulation of OKT9 125I-Fab fragments indicated that diferric transferrin caused a marked decrease in the amount of internalized 125I-Fab fragments associated with the cells after 60 min of incubation at 37 degrees C. Diferric transferrin therefore increases the efficiency of the release of internalized 125I-Fab fragments compared with cells incubated without diferric transferrin. These data indicate that transferrin regulates the sorting of the transferrin receptor at the cell surface and within endosomal membrane compartments.

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Inhibition of the receptor-mediated endocytosis of diferric transferrin is associated with the covalent modification of the transferrin receptor with palmitic acid.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(17)46270-3 ◽

1990 ◽

Vol 265 (27) ◽

pp. 16644-16655 ◽

Cited By ~ 8

Author(s):

E Alvarez ◽

N Gironès ◽

R J Davis

Keyword(s):

Palmitic Acid ◽

Transferrin Receptor ◽

Covalent Modification ◽

Receptor Mediated Endocytosis ◽

Diferric Transferrin

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Receptor-Mediated Recognition and Uptake of Iron from Human Transferrin by Staphylococcus aureus andStaphylococcus epidermidis

Infection and Immunity ◽

10.1128/iai.66.8.3591-3596.1998 ◽

1998 ◽

Vol 66 (8) ◽

pp. 3591-3596 ◽

Cited By ~ 34

Author(s):

Belinda Modun ◽

Robert W. Evans ◽

Christopher L. Joannou ◽

Paul Williams

Keyword(s):

Staphylococcus Aureus ◽

Transferrin Receptor ◽

Polyacrylamide Gel Electrophoresis ◽

Surface Protein ◽

Binding Assays ◽

Human Transferrin ◽

Protein Receptor ◽

Iron Deficient ◽

Competition Binding ◽

Diferric Transferrin

ABSTRACT Staphylococcus aureus and Staphylococcus epidermidis both recognize and bind the human iron-transporting glycoprotein, transferrin, via a 42-kDa cell surface protein receptor. In an iron-deficient medium, staphylococcal growth can be promoted by the addition of human diferric transferrin but not human apotransferrin. To determine whether the staphylococcal transferrin receptor is involved in the removal of iron from transferrin, we employed 6 M urea–polyacrylamide gel electrophoresis, which separates human transferrin into four forms (diferric, monoferric N-lobe, and monoferric C-lobe transferrin and apotransferrin). S. aureus and S. epidermidis but notStaphylococcus saprophyticus (which lacks the transferrin receptor) converted diferric human transferrin into its apotransferrin form within 30 min. During conversion, iron was removed sequentially from the N lobe and then from the C lobe. Metabolic poisons such as sodium azide and nigericin inhibited the release of iron from human transferrin, indicating that it is an energy-requiring process. To demonstrate that this process is receptor rather than siderophore mediated, we incubated (i) washed staphylococcal cells and (ii) the staphylococcal siderophore, staphyloferrin A, with porcine transferrin, a transferrin species which does not bind to the staphylococcal receptor. While staphyloferrin A removed iron from both human and porcine transferrins, neither S. aureus nor S. epidermidis cells could promote the release of iron from porcine transferrin. In competition binding assays, both native and recombinant N-lobe fragments of human transferrin as well as a naturally occurring human transferrin variant with a mutation in the C-lobe blocked binding of 125I-labelled transferrin. Furthermore, the staphylococci removed iron efficiently from the iron-loaded N-lobe fragment of human transferrin. These data demonstrate that the staphylococci efficiently remove iron from transferrin via a receptor-mediated process and provide evidence to suggest that there is a primary receptor recognition site on the N-lobe of human transferrin.

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Transcriptional regulation by iron of the gene for the transferrin receptor

Molecular and Cellular Biology ◽

10.1128/mcb.6.1.236-240.1986 ◽

1986 ◽

Vol 6 (1) ◽

pp. 236-240

Author(s):

K Rao ◽

J B Harford ◽

T Rouault ◽

A McClelland ◽

F H Ruddle ◽

...

Keyword(s):

Transferrin Receptor ◽

K562 Cells ◽

Iron Chelator ◽

Transcriptional Level ◽

Transferrin Receptors ◽

Intracellular Iron ◽

Human Transferrin Receptor ◽

Diferric Transferrin ◽

Permeable Iron

Treatment of K562 cells with desferrioxamine, a permeable iron chelator, led to an increase in the number of transferrin receptors. Increasing intracellular iron levels by treatment of cells with either human diferric transferrin or hemin lowered the level of the transferrin receptors. By using a cDNA clone of the human transferrin receptor, we showed that the changes in the levels of the receptor by iron were accompanied by alterations in the levels of the mRNA for the receptor. The rapidity of these changes indicated that the mRNA had a very short half-life. By using an in vitro transcriptional assay with isolated nuclei, we obtained evidence that this regulation occurred at the transcriptional level.

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